A novel cyclic NP1 reveals obstruction of EGFR kinase activity and attenuation of EGFR-driven cell lines

Aberrations of the epidermal growth factor receptor (EGFR), for example, mutations and overexpression, play pivotal roles in various cellular functions, such as proliferation, migration, and cell differentiation. Approved small molecule-based inhibitors, including gefitinib and erlotinib, are used clinically to target the tyrosine kinase domain of EGFR (TK-EGFR). However, the severity of the side effects, off-target effects, and drug resistance is a concern. Cyclic peptides are a well-known peptide format with high stability and are promising molecules for drug development. Herein, the Ph.D.(TM)-C7C phage display library was used to screen cyclic peptides against TK-EGFR. Biopanning, both with and without propagation methods, was performed to assess the highest capacity peptides using the enzymatic activity of TK-EGFR. Interestingly, NP1, a peptide selected during biopanning without propagation demonstrated an inhibitory effect against TK-EGFR at IC50 within the nanomolar range; this effect was better than that of P1 obtained using biopanning with propagation. Moreover, NP1 elicited EGFR with an affinity binding (K-D) value of 18.40 +/- 5.50 mu M by surface plasmon resonance (SPR). Introducing cell-penetrating peptides or Arginine-9 (Arg9) at the N-terminus of NP1 thus improves cell-penetrability and can lead to the inhibition of EGFR-driven cancer cell lines; however, it exhibits no hepatotoxicity. Furthermore, NP1 caused a decrease in phosphorylated EGFR after activation within cells. A docking model shows that NP1 interacted primarily with TK-EGFR via hydrogen bonding. Together, this suggests that NP1 is a novel EGFR peptide inhibitor candidate with specificity and selectivity toward TK-EGFR, and may be applied to targeted therapy.